Stable and soluble antibodies inhibiting TNFα

ABSTRACT

The present invention relates to particularly stable and soluble scFv antibodies and Fab fragments specific for TNFα, which comprise specific light chain and heavy chain sequences that are optimized for stability, solubility, in vitro and in vivo binding of TNFα, and low immunogenicity. Said antibodies are designed for the diagnosis and/or treatment of TNFα-related disorders. The nucleic acids, vectors and host cells for expression of the recombinant antibodies of the invention, methods for isolating them and the use of said antibodies in medicine are also disclosed.

The present application is a continuation of U.S. patent applicationSer. No. 14/185,783 filed Feb. 20, 2014 (now allowed); which is adivisional of U.S. application Ser. No. 13/708,500 filed Dec. 7, 2012(now U.S. Pat. No. 8,691,228) which is a divisional of U.S. applicationSer. No. 13/245,420 filed Sep. 26, 2011 (now U.S. Pat. No. 8,389,693);which is a continuation of U.S. application Ser. No. 11/916,793 filedMay 27, 2008 (now U.S. Pat. No. 8,067,547), which is the National Stageof International Application Serial No.: PCT/CH2006/000300 filed Jun. 6,2006, which claims benefit to U.S. Provisional Patent Application Ser.Nos. 60/785,353, filed Mar. 23, 2006, and 60/687,971 filed Jun. 7, 2005.

TECHNICAL FIELD

The present invention relates to optimised antibodies and antibodyderivatives that bind to and block the function of tumour necrosisfactor alpha (TNFα) and are useful for the diagnosis and/or treatment,prevention or amelioration of TNFα-associated diseases; their codingsequences, production, and use in pharmacologically suitablecompositions.

BACKGROUND ART

Tumour necrosis factor alpha (TNFα, also known as cachectin), is anaturally occurring mammalian cytokine produced by numerous cell types,including monocytes and macrophages in response to endotoxin or otherstimuli. TNFα is a major mediator of inflammatory, immunological, andpathophysiological reactions (Grell, M., et al. (1995) Cell, 83:793-802).

Soluble TNFα is formed by the cleavage of a precursor transmembraneprotein (Kriegler, et al. (1988) Cell 53: 45-53), and the secreted 17kDa polypeptides assemble to soluble homotrimer complexes (Smith, et al.(1987), J. Biol. Chem. 262: 6951-6954; for reviews of TNF, see Butler,et al. (1986), Nature 320:584; Old (1986), Science 230: 630). Thesecomplexes then bind to receptors found on a variety of cells. Bindingproduces an array of pro-inflammatory effects, including (i) release ofother pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, andIL-1, (ii) release of matrix metalloproteinases and (iii) up regulationof the expression of endothelial adhesion molecules, further amplifyingthe inflammatory and immune cascade by attracting leukocytes intoextravascular tissues.

A large number of disorders are associated with elevated levels of TNFα,many of them of significant medical importance. TNFα has been shown tobe up-regulated in a number of human diseases, including chronicdiseases such as rheumatoid arthritis (RA), inflammatory bowel disordersincluding Crohn's disease and ulcerative colitis, sepsis, congestiveheart failure, asthma bronchiale and multiple sclerosis. Mice transgenicfor human TNFα produce high levels of TNFα constitutively and develop aspontaneous, destructive polyarthritis resembling RA (Keffer et al.1991, EMBO J., 10, 4025-4031). TNFα is therefore referred to as apro-inflammatory cytokine.

TNFα is now well established as key in the pathogenesis of RA, which isa chronic, progressive and debilitating disease characterised bypolyarticular joint inflammation and destruction, with systemic symptomsof fever and malaise and fatigue. RA also leads to chronic synovialinflammation, with frequent progression to articular cartilage and bonedestruction. Increased levels of TNFα are found in both the synovialfluid and peripheral blood of patients suffering from RA. When TNFαblocking agents are administered to patients suffering from RA, theyreduce inflammation, improve symptoms and retard joint damage (McKown etal. (1999), Arthritis Rheum. 42:1204-1208).

Physiologically, TNFα is also associated with protection from particularinfections (Cerami. et al. (1988), Immunol. Today 9:28). TNFα isreleased by macrophages that have been activated by lipopolysaccharidesof Gram-negative bacteria. As such, TNFα appears to be an endogenousmediator of central importance involved in the development andpathogenesis of endotoxic shock associated with bacterial sepsis(Michie, et al. (1989), Br. J. Surg. 76:670-671.; Debets. et al. (1989),Second Vienna Shock Forum, p. 463-466; Simpson, et al. (1989) Crit. CareClin. 5: 27-47; Waage et al. (1987). Lancet 1: 355-357; Hammerle. et al.(1989) Second Vienna Shock Forum p. 715-718; Debets. et al. (1989),Crit. Care Med. 17:489-497; Calandra. et al. (1990), J. Infect. Dis.161:982-987; Revhaug et al. (1988), Arch. Surg. 123:162-170).

As with other organ systems, TNFα has also been shown to play a key rolein the central nervous system, in particular in inflammatory andautoimmune disorders of the nervous system, including multiplesclerosis, Guillain-Barre syndrome and myasthenia gravis, and indegenerative disorders of the nervous system, including Alzheimer'sdisease, Parkinson's disease and Huntington's disease. TNFα is alsoinvolved in disorders of related systems of the retina and of muscle,including optic neuritis, macular degeneration, diabetic retinopathy,dermatomyositis, amyotrophic lateral sclerosis, and muscular dystrophy,as well as in injuries to the nervous system, including traumatic braininjury, acute spinal cord injury, and stroke.

Hepatitis is another TNFα-related inflammatory disorder which amongother triggers can be caused by viral infections, includingEpstein-Barr, cytomegalo-virus, and hepatitis A-E viruses. Hepatitiscauses acute liver inflammation in the portal and lobular region,followed by fibrosis and tumor progression.

TNFα can mediate cachexia in cancer, which causes most cancer morbidityand mortality (Tisdale M. J. (2004), Langenbecks Arch Surg.389:299-305).

The key role played by TNFα in inflammation, cellular immune responsesand the pathology of many diseases has led to the search for antagonistsof TNFα.

TNFα is an important cytokine whose systemic blockade carries the riskfor increased frequency and severity of clinically manifestedinfections, in particular re-activation of latent tuberculosis andpossibly other risks including induction of lymphomas, demyelinatingdiseases and heart failure.

One class of TNFα antagonists designed for the treatment ofTNFα-mediated diseases are antibodies or antibody fragments thatspecifically bind TNFα and thereby block its function. The use ofanti-TNFα antibodies has shown that a blockade of TNFα can reverseeffects attributed to TNFα including decreases in IL-1, GM-CSF, IL-6,IL-8, adhesion molecules and tissue destruction (Feldmann et al. (1997),Adv. Immunol. 1997:283-350).

Antibodies directed against TNFα have been proposed for the prophylaxisand treatment of endotoxic shock (Beutler et al. (1985) Science: 234,470-474). The use of anti-TNFα antibodies in the treatment of septicshock is discussed by Bodmer et al., 1993, (Critical Care Medicine,21:441-446, 1993), Wherry et al., 1993, (Critical Care Medicine,21:436-440) and Kirschenbaum et al., 1998, (Critical Care Medicine,26:1625-1626).

A method for treating a neurodegenerative disease in a human byadministering an anti-TNFα monoclonal antibody or a TNFα bindingfragment thereof has been disclosed in US2003147891.

WO0149321 teaches the use of TNFα blockers including anti TNFαantibodies to treat neurologic and related disorders caused by TNFα. Itprovides a method for treating said disorders by administering a TNFαantagonist.

WO03047510 discloses various kinds of monoclonal and engineeredantibodies directed against TNFα, their production, compounds comprisingthem and use in medicine.

Antibodies useful for therapies of TNFα mediated diseases are usuallyeither monoclonal antibodies (mAB) produced by hybridoma technology froma natural source, usually a mouse, or engineered antibodies. The lattereither correspond to naturally occurring antibodies in that theycomprise full-length heavy and light chains, or to the Fab fragmentsthat can also be generated from natural antibodies by proteolyticcleavage, or to single chain scFv antibodies wherein fragments of thevariable heavy and light chain regions are linked by a peptide linker.

Both, heavy and light chains of an antibody comprise constant andvariable domains. As non-human antibodies are immunogenic, the amount ofhuman-like sequences in an antibody is often increased in a so-called“hybrid” antibody, which comprises constant regions of a human IgG, andvariable regions matching the sequences of an animal antibody, in mostcases murine antibodies with the desired specificity. These variableregions can then be further adapted to become more similar to a typicalhuman antibody by mutagenesis, leading to a “humanised” antibody. In yetan alternative approach, only the antigen binding portions, i.e. thecomplementary determining regions (CDRs) of the variable regions of amouse antibody are combined with a framework of a human antibody,resulting in a “CDR-grafted” antibody.

Monoclonal antibodies against TNFα have been described in the prior art.Meager et al., 1087 (Hybridoma 6:305-311) describe murine monoclonalantibodies against recombinant TNFα. Shimamoto et al., 1988, (ImmunologyLetters 17:311-318) describe the use of murine monoclonal antibodiesagainst TNFα in preventing endotoxic shock in mice.

U.S. Pat. No. 5,919,452 discloses anti-TNFα chimeric antibodies andtheir use in treating pathologies associated with the presence of TNFα.

The use of anti-TNFα antibodies in the treatment of RA and Crohn'sdisease is discussed in Feldman et al. (1998), (TransplantationProceedings 30:4126-4127), Adorini et al., 1997, (Trends in ImmunologyToday 18:209-211) and in Feldmann et al., 1997, (Advanced Immunology64:283-350). The antibodies to TNFα used in such treatments aregenerally chimeric antibodies, such as those described in U.S. Pat. No.5,919,452.

US20003187231 discloses humanised anti-TNFα antibodies with at least onenon-human CDR region that have improved binding characteristics.Furthermore, in the International Patent Application WO 92/11383,recombinant antibodies, including CDR-grafted antibodies, specific forTNFα are disclosed. Rankin et al. (1995), (British J. Rheumatology34:334-342) describe the use of such CDR-grafted antibodies in thetreatment of RA.

WO9211383 discloses a recombinant, humanised CDR-grafted antibodyspecific for TNFα that is derived from the murine monoclonal antibody61E7, hTNFI, hTNF3 or 101.4, and it teaches the production and use ofsaid antibodies in diagnosis and/or therapy of TNFα-associateddisorders.

Among the specific inhibitors of TNFα that have become commerciallyavailable only recently, a monoclonal, chimeric mouse-human antibodydirected against TNFα (infliximab, Remicade™; CentocorCorporation/Johnson & Johnson) has demonstrated clinical efficacy in thetreatment of RA (Elliott et al. 1994, Lancet 344:1105-1110; Mani et al.(1998), Arthritis & Rheumatism 41: 1552-1563). Infliximab has alsodemonstrated clinical efficacy in the treatment of the inflammatorybowel disorder Crohn's disease (Baert et al. 1999, Gastroenterology 116:22-28.)

US22002037934 discloses the treatment of hepatitis by administration ofan anti-TNFα antibody such as infliximab.

U.S. Pat. No. 6,428,787 teaches the treatment of neurologic andTNFα-associated diseases with anti-TNFα antibodies including infliximab,CDP571 and D2E7.

D2E7 (Adalimumab), a human anti-TNFα monoclonal antibody (Abbott) hasbeen developed to treat RA and Crohn's disease (WO9729131). Celltech isdeveloping CDP571 (EP0626389), a humanised monoclonal anti-TNFα IgG4antibody to treat Crohn's disease and CDP870, a humanised monoclonalanti TNFα antibody fragment to treat RA. The local administration ofsaid antibodies for treatment of localised disorders is disclosed inUS2003185826.

Many single chain antibodies (scFvs) were generated against a multitudeof different antigens, in particular because they can be easily selectedfor high binding capacity using techniques such as for example phagedisplay or ribosome display. Moreover, scFv antibodies can be producedin microbial systems which are associated with fewer costs compared tothe production of therapeutic full-length antibodies.

In addition to conventional extracellular and in vitro applications,scFvs have also been successfully used for intracellular applications(Wörn et al. 2000, JBC, 28; 275(4):2795-2803; Auf der Maur et al. 2002,JBC, 22; 277(47):45075-45085; Stocks MR, 2004, Drug Discov Today. 15;9(22):960-966); hence, scFvs directed against intracellular antigenshave been developed. In general, intracellular expression of functionalscFvs is limited by their instability, insolubility, and tendency toform aggregates. For this reason, in vivo screening systems for scFvantibodies, which are particularly soluble and stable under reducingconditions typical for the intracellular environment (e.g. nucleus,cytoplasm) have been successfully developed using a so called “QualityControl” screen (WO0148017; Auf der Maur et al. (2001), FEBS Lett.508:407-412; Auf der Maur et al. (2004), Methods 34:215-224) and haveled to the identification of particularly stable and soluble scFvframework sequences for such purposes (WO03097697). Furthermore, theseframeworks show exceptional expression levels and enhanced stability andsolubility properties also under natural, oxidizing conditions in theextracellular environment. Hence, these favourable biophysical andbiochemical properties translate into favourable high production yieldsand enable these antibody fragments, once directed against specificantigens, to be applied locally and/or systemically as proteintherapeutics in particular therapeutic areas. As both scFv antibodiesand Fab fragments, in contrast to full-length antibodies, lack the Fcpart that is recognized by the Fc-receptor of monocytes, such as e.g.natural killer cells, they do not evoke antibody-dependent cell-mediatedcytotoxicity (AD CC) and thus do not provoke unspecific toxicity due tobinding to Fc-receptors on non-target cells.

Hence, there is a need for new, effective forms of antibodies for thetreatment for TNFα-associated disorders such as RA, particularlytreatments that can provide sustained, controlled therapy by localadministration with a low degree of side effects. The present inventionprovides antibodies, compositions and methods for effective andcontinuous treatment of inflammatory processes of arthritis and otherTNFα-mediated disorders or pathophysiological mechanisms, in particularvarious forms of pain.

All publications and references cited herein are hereby incorporated byreference in their entirety.

DISCLOSURE OF THE INVENTION

Hence, it is a general object of the invention to provide a stable andsoluble antibody or antibody derivative, which specifically binds TNFαin vitro and in vivo. In a preferred embodiment said antibody derivativeis an scFv antibody or Fab fragment.

Now, in order to implement these and still further objects of theinvention, which will become more readily apparent as the descriptionproceeds, said antibody or antibody derivative is manifested by thefeatures that it comprises a light chain variable domain being orderived from the sequence SEQ ID NO:1 that is combined with a heavychain variable domain being or derived from the sequence SEQ ID NO:2,wherein in the case of a derived sequence said sequence has at maximumup to 5 changes within the framework of said VL domain and/or at maximumup to 9 changes within the framework of said VH domain.

A preferred embodiment of the present invention is said antibody orantibody derivative, wherein one or more amino acid changes areintroduced at any of the positions in the framework, preferably at oneor more positions selected from the group of the positions 4, 46, 65, 6770, and 83 of the VL domain, and/or at one or more of the positionsselected from the group of the positions 11, 16, 28, 43, 48, 68, 70, 71,72, 73, 76, 77, 79, 93 and 112 of the VH domain. More preferably, atleast one of the conversions leads to an amino acid present in SEQ IDNO:3 for VL and/or SEQ ID NO:4 for VH, and even more preferably at most13 conversions in total are present.

Most preferably, said antibody or antibody derivatives comprises a VLdomain of the sequence SEQ ID NO:1 and/or a VH domain of the sequence orderived from the sequence SEQ ID NO:2, or a VL domain of the sequenceSEQ ID NO:11 and a VH domain of the sequence SEQ ID NO:4. If the VHdomain of the antibody of the present invention comprises a VL domain ofSEQ ID NO:1, in a preferred embodiment the VH sequence is derived fromSEQ ID NO:2 such that the phenylalanine at position 68 is changed toeither alanine, leucine, isoleucine or valine. Additional changes withinVH are optional. scFv antibodies of this kind are given in SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ IDNO:37.

In another preferred embodiment of the present invention said antibodyor antibody derivative is derived from the antibody with the VL sequenceSEQ ID NO:1 and the VH sequence SEQ ID NO:2 and comprises at least oneamino acid residue that is converted in at least one of the CDRs to aresidue present in the corresponding CDR of the VL sequence SEQ ID NO:5and/or the VH sequence SEQ ID No:6 or SEQ ID NO:25.

In a much preferred embodiment of the present invention in said antibodyor antibody derivative at least one of the CDRs of the group VL CDR2, VLCDR3, VH CDR2 or VH CDR3 is converted to the corresponding CDR of the VLsequence SEQ ID NO:5 and/or the VH sequence SEQ ID No:25 or SEQ ID NO:6.

Most preferably, said antibody or antibody derivative comprises thefollowing VL/VH sequence combinations:

-   VL SEQ ID NO:7/VH SEQ ID NO:2,-   VL SEQ ID NO:8/VH SEQ ID NO:2,-   VL SEQ ID NO:1/VH SEQ ID NO:9,-   VL SEQ ID NO:1/VH SEQ ID NO:25,-   VL SEQ ID NO:1/VH SEQ ID NO:28,-   VL SEQ ID NO:1/VH SEQ ID NO:29,-   VL SEQ ID NO:26/VH SEQ ID NO:30,-   VL SEQ ID NO:27/VH SEQ ID NO:30.

In another preferred embodiment the antibody or antibody derivative ofthe present invention has specificity to human TNFα. Preferably, antigenbinding is characterized by a K_(d) of ≈100 nM or less. More preferredis an antibody with a K_(d) of 10 nM or less, and most preferred of 1 nMand less.

Antibody derivatives according to the present invention are for exampleFc fusions, toxin fusions, fusions to enzymatic activities, differentformats such as minibodies, diabodies, linear antibodies, single chainantibodies, bispecific antibody fragments, in particular scFv and Fabfragments.

Another preferred object of the present invention is a scFv antibodywhose VL and VH domains are connected by a linker, preferably in aVL-linker-VH sequence arrangement. More preferably said linker has thesequence SEQ ID NO: 10.

Another preferred object of the present invention is a scFv antibodyderived from SEQ ID NO:40 (TB-A). Such an antibody can be obtained bymutagenesis, and comprises three or less mutations in either framework,CDR and/or linker sequences. Preferably, the scFv antibody has thesequence SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

Another preferred object of the present invention is the Fab fragmentcomprising a VL domain that is fused to the constant region of a humanIg kappa chain, and a VH domain that is fused to the CH1 domain of ahuman IgG, whereby the two fusion polypeptides are connected by aninter-chain disulfide bridge.

Still in another aspect the antibody or antibody derivative, e.g. theantibody fragment, of the present invention is labelled or chemicallymodified.

The present invention also provides a DNA sequence encoding any of theantibodies or antibody derivatives of the present invention, as well asa cloning or expression vector containing said DNA sequence. Inaddition, a suitable host cell transformed with said DNA sequence isprovided, which preferentially is E. coli, a yeast or a mammalian cell.

Furthermore, a method for the production of the antibodies or antibodyderivatives of the present invention is provided, comprising culturingof the host cell transformed with the DNA encoding any of saidantibodies or antibody derivatives under conditions that allow thesynthesis of said antibody or antibody derivative, and recovering saidmolecule from said culture. Preferably, said method provides an scFvantibody or Fab fragment purified from E. coli.

Another aspect of the present invention is the use of the antibodies orantibody derivatives provided by the present invention as a diagnostictool for in vitro diagnostics, and/or as a pharmaceutical. This use isparticularly preferred in the context of any TNFα related condition.

The present invention also encompasses a composition comprising anantibody or antibody derivative of the present invention in combinationwith a pharmaceutically acceptable carrier, diluent or excipient, saidcomposition to be used as a medicament for the treatment of TNFαassociated diseases.

In a further aspect the present invention provides a combinationpreparation comprising an antibody or antibody derivative of the presentinvention, preferably with a second compound that is not an antibody orantibody derivative specific for TNFα.

In yet another aspect of the present invention the vector comprising theDNA sequence encoding an scFv antibody of the present invention is usedfor gene therapy.

The treatment of TNFα associated diseases is achieved by blocking ofTNFα due to a strong interaction of TNFα with the antibody or theantibody derivative. Preferably, a treatment of autoimmune, acute orchronic inflammation conditions, cancer-related diseases, pain,neurological and neurodegenerative disorders, infectious diseases andcardiovascular diseases is envisaged.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing de-tailed description thereof. Such description makesreference to the annexed drawings, wherein:

FIG. 1 shows a scheme of the scFv antibodies with the sequences of TB-Aand TB-B delimiting the range of the most frequent variations. Asterisksdesignate positions at which amino acid changes in the framework of theantibodies of the present invention are tolerated. Amino acids indicatedbelow the CDRs (the CDRs being emphasized with a gray background) can beused in the respective CDRs. TB-A VL is SEQ ID NO: 1; TB-A VH is SEQ IDNO: 2; TB-B VL is SEQ ID NO: 3; TB-B VH is SEQ ID NO: 4; the linkersequence (GGGGS)₄ is SEQ ID NO: 10.

FIG. 2 shows an exemplary scheme for the expression of a Fab fragment.

FIGS. 3A and 3B show the production yield of scFvs when expressed in E.coli. FIG. 3A shows SDS-polyacryalmide gel electrophoresis of expressedproteins. FIG. 3B shows analytical gel filtration of TB-A and TB-wtshowing superior solubility of TB-A.

FIGS. 4A and 4B show shows a comparison of affinity of different scFvantibodies towards TNFα determined by ELISA.

FIGS. 5A, 5B, and 5C show the inhibition of human TNFα-inducedcytotoxicity in mouse L929 fibroblasts. FIG. SA shows concentrationdependent inhibition of TB-A in comparison to TB-wt and infliximab withIC₅₀ values. FIG. 5B shows comparison of TB-A derivatives to block TNFαinduced cytotoxicity. FIG. 5C shows comparison of scFv and Fab formatsof TB-A.

FIG. 6 shows the effect of antibody treatment of human TNFα-inducedjoint swelling in rat (Experiment: 5.3., Experiment 1).

FIG. 7 shows the scoring scheme for histopathological inflammationscoring.

FIG. 8 shows the effect of antibody treatment on human TNFα-inducedjoint inflammation in rat (Experiment: 5.3., Experiment 1).

FIG. 9 shows the effect of antibody treatment of human TNFα-inducedjoint swelling in rat (Experiment: 5.3., Experiment 2).

FIG. 10 shows the effect of antibody treatment on human TNFα-inducedjoint inflammation in rat (Experiment: 5.3., Experiment 2).

FIG. 11 shows the stability of TB-A in different body fluids.

MODES FOR CARRYING OUT THE INVENTION

It has been found that antibodies or antibody derivatives comprising theframeworks identified in the so called “quality control” screen(WO0148017) are characterised by a generally high stability and/orsolubility and thus may also be useful in the context of extracellularapplications such as neutralizing TNFα. The present invention providesantibodies or antibody derivatives characterized by enhanced stabilityand solubility that specifically recognize and bind TNFα and thus aresuitable to block the function of TNFα in vivo. Said antibodies orantibody derivatives are characterized by a special framework derivedfrom the “quality control” screen for antibodies with particularlystable and soluble frameworks independent of their antigen binding sitethat has been disclosed in EP1479694. If the frameworks used in thescreening are human antibody frameworks, they can be considered asnon-immunogenic frameworks for human applications. The CDRs of theantibodies of the present invention are identical to or derived from theCDRs of the murine monoclonal antibody Di62 (Döring et al., 1994) thatspecifically binds to human TNFα with a high affinity (Kd=0.4 nM) andcan block TNFα binding to its receptor. In addition, Di62 inhibits humanTNFα-induced cytotoxicity in mouse L929 cells. The obvious step ofgrafting the CDRs from the mouse antibody onto the apparently bestsuitable human acceptor framework with undefined antigen bindingproperties, said framework having the VL sequence SEQ ID NO:5 and the VHsequence SEQ ID NO:6, said sequences being linked by a (GGGGS)₄ linker(SEQ ID NO:10), resulted in an scFv antibody of the sequence

(SEQ ID NO: 3 + SEQ ID NO: 10 + SEQ ID NO: 4)DIVLTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKRLIYSAFNRYTGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYSFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS said scFv antibody being called TB-B. TB-B gave good yields in proteinexpression (FIG. 3a ), but was unable to specifically bind TNFα (FIG. 4a).

Hence, to obtain an antibody or antibody derivative that is (i)sufficiently specific for binding TNFα, (ii) sufficiently soluble toallow efficient production and purification and to block TNFα in vivo,(iii) sufficiently stable to be useful as a pharmaceutical withoutsuffering a rapid degradation and (iv) sufficiently non-immunogenic, acompromise between best solubility and best antigen bindingcharacteristics was sought by varying the framework and the CDRs. Thepresent invention provides a sequence for VL and VH that is optimizedfor the combination of the criteria (i-iv). An scFv antibody comprisingsaid VL (SEQ ID. NO:1 linked by a (GGGGS)₄ (SEQ ID NO: 10) linker tosaid VH (SEQ ID NO:2) is called TB-A. The sequence of TB-A is given bySEQ ID NO:40. This antibody is still reasonably stable and soluble togive satisfactory yields when expressed and purified from E. coli (FIG.3A), and it does not aggregate (FIG. 3B). Its binding characteristicstowards TNFα are excellent, with a Kd of 0.8 nM.

The present invention also discloses VL and VH sequences derived fromthe sequences present in TB-A in various ways. First, point mutations atup to five positions in the framework of VL and/or at up to ninepositions in the framework of VH have been found acceptable, especiallypoint mutations that render the frameworks more TB-B-like, i.e. morelike SEQ ID NO:3 for VL or SEQ ID NO:4 for VH. An scFv antibodycomprising a VL domain of the sequence SEQ ID NO:11 and a VH domain ofthe sequence SEQ ID NO:4, linked by the (GGGGS)₄ linker (SEQ ID NO: 10),is called TB-B R46L because it differs only at position 46 of VL fromTB-B. In contrast to TB-B this antibody still has good bindingproperties towards TNFα (Kd≈100 nM). This suggests that the number ofchanges in TB-B R46L relative to TB-A represents approximately the upperlimit for changes in the variable domain framework.

In a preferred embodiment of the present invention only single or doublepoint mutations are introduced into VL and/or VH frameworks of TB-A. Thepreferred framework residues for mutations are at positions 4, 46, 65,67, 70 and 83 for VL, and at positions 11, 16, 28, 43, 48, 68, 70, 71,72, 73, 76, 77, 79, 93 and 112 for VH. The positions are numberedaccording to the numbering in the sequence listings. The amino acidssubstitutions are preferably either “conservative”, or such that thereplacing amino acids are more similar or preferably even identical tothe corresponding amino acids present in the TB-B sequence. For example,A76 of VH in TB-A can be changed to 176 as it is present in TB-B, but itmay also be changed to another amino acid with similar, i.e. a non-polarside chain such as V or L. This is an example of a “conservative” aminoacid substitution. Families of amino acid residues having similar sidechains suitable for “conservative” substitutions as used herein havebeen defined in the art, including basic side chains (K, R, H), acidicside chains (D, E), uncharged polar side chains (Q, N, S, T, Y, C),non-polar side chains (G, A, V, L, I, P, F, M, W), beta-branched sidechains (T, V, I) and aromatic side chains (Y, F, W, H). A preferredconservative change is that of VL at position 83, in that V is changedto either F (SEQ ID NO:26) or to A (SEQ ID NO: 27). However, in SEQ IDNO: 32 a non-conservative change in VL is V83E, which is combined with achange in CDR1, i.e. N31D, and in VH with V79A. Another extraordinaryTB-A variant is that of SEQ ID NO: 33, with a conservative F68L exchangein VH connected to VL by a linker carrying an R at position 2, replacingG.

Much preferred single amino acid exchanges are R655 or Y67S in VL andK43Q or F68 to V, L, or A in VH. Much preferred double changes areF70L/L72R or A76I/S77G in VH. ScFv antibodies comprising TB-A sequenceswith said alterations show inhibition of TNFα induced cytotoxicity inL929 cells. The results of some of them are shown in FIG. 5B. Theirsequences are as follows:

-   SEQ ID NO:18=TB-A H_K43Q (TB-A H43)-   SEQ ID NO:19=TB-A H_F68V (TB-A H68)-   SEQ ID NO:20=TB-A H_F70L/L72R (TB-A H70/72)-   SEQ ID NO:21=TB-A H_A76I/S77G (TB-A H76/77)-   SEQ ID NO:22=TB-A L_L46R (TB-A L46)-   SEQ ID NO:23=TB-A L_R65S (TB-A L65)-   SEQ ID NO:24=TB-A L_Y67S (TB-A L67)

In a preferred embodiment any of the above mentioned VH domains may becombined with any of the above mentioned VL domains.

In another preferred embodiment of the present invention the VL and VHdomains of TB-A and TB-B are shuffled such that the VL domain of TB-A(SEQ ID. NO:1 is combined with the VH domain of TB-B (SEQ ID NO:4), orthe VL domain of TB-B (SEQ ID. NO:4) is combined with the VH domain ofTB-A (SEQ ID NO:2). In a much preferred embodiment the shuffled versionsobtained in an scFv are connected with the (GGGGS)₄ linker of thesequence SEQ ID. NO:10, resulting in the scFv antibodies TB-AB (SEQ ID.NO:12) or TB-BA (SEQ ID. NO:13), respectively. Said (GGGGS)₄ (SEQ ID NO:10) linker can have an amino acid exchange of a glycine to a morehydrophilic, i.e. polar, or even charged amino acid, which may renderthe antibody more soluble. Among these variations, the one with thesequence GRGGS-(GGGGS)3 (SEQ ID NO:39) is preferred.

It is also within the scope of the present invention to combine VL or VHdomains of TB-B-like sequences with VH or VL domains of TB-A-likesequences, whereby TB-B/TB-A-like means that the sequences are closer tothe one than to the other.

In yet another preferred embodiment of the present invention one or moreamino acids are changed in the CDR regions of the TB-A VL and/or VHsequences to match the corresponding amino acids present in the selectedsequences SEQ ID NO:5 of VL and/or SEQ ID. NO:6 or SEQ ID NO:25 of VH.Much preferred are changes in one of the VL CDRs (VL CDR2 or VL CDR3)and/or VH CDRs (CDR2 or CDR3), with the most preferred changes leadingto the VL sequences SEQ ID NO:7 or SEQ ID NO:8 and/or the VH sequencesSEQ ID NO:25 or SEQ ID NO:9, respectively.

In another preferred embodiment of the present invention the VLsequences SEQ ID NO:26 or SEQ ID NO:27 is combined with the VH sequenceSEQ ID NO:30. In yet another preferred embodiment of the presentinvention the VL sequence SEQ ID NO:1 is combined with the VH sequencesSeq ID NO:28 or SEQ ID NO:29. Generally, any of the disclosed VLsequences may be combined with any of the disclosed VH sequences.

Objects of the present invention are antibodies and antibody fragments,in particular VL or VH polypeptides, single-chain antibodies (scFv) orFab fragments. In the case of scFv antibodies, a selected VL domain canbe linked to a selected VH domain in either orientation by a flexiblelinker. A suitable state of the art linker consists of repeated GGGGS(SEQ ID NO: 41) amino acid sequences or variants thereof. In a preferredembodiment of the present invention a (GGGGS)₄ linker (SEQ ID NO:10) orits derivative SEQ ID NO: 39 is used, but variants of 1-3 repeats arealso possible (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90:6444-6448). Other linkers that can be used for the present inventionare described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi etal. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 andRoovers et al. (2001), Cancer Immunol. Immunother. 50:51-59. Thearrangement can be either VL-linker-VH or VH-linker-VL, with the formerorientation being the preferred one.

In the case of Fab fragments, selected light chain variable domains VLare fused to the constant region of a human Ig kappa chain, while thesuitable heavy chain variable domains VH are fused to the first(N-terminal) constant domain CH1 of a human IgG. In an exemplaryembodiment of the present invention, the human Cκ. domain has thesequence SEQ ID NO:14 and the CH1 domain used to construct the Fabfragments has the sequence SEQ ID NO:15. FIG. 2 shows an example of aFab fragment wherein the VL and VH domains of TB-A are used such thatthe VL domain is directly linked to the human kappa constant domain,resulting in the sequence

(SEQ ID NO: 1 + SEQ ID NO: 14)DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGECand the VH domain is fused to the first constant domain (CH1), resultingin the sequence

(SEQ ID NO: 2 + SEQ ID NO: 15)QVQLVQSGAEVKKPGASVINSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFSEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCTS.

At the C-terminus, an inter-chain disulfide bridge is formed between thetwo constant domains.

The antibodies or antibody derivatives of the present invention can haveaffinities to human TNFα with dissociation constants K_(d) in a range of0.8-10,000 nM. In a preferred embodiment of the present invention theK_(d) is ≦10 nM. The affinity of an antibody for an antigen can bedetermined experimentally using a suitable method (Berzofsky et al.“Antibody-Antigen Interactions”, in Fundamental Immunology, Paul, W. E.,Ed, Raven Press: New York, N.Y. (1992); Kuby, J. Immunology, W.H.Freeman and Company: New York, N.Y.) and methods described therein.

In one aspect of the present invention the antibodies or antibodyderivatives, especially the scFv or Fab fragments, are labeled.Detectable labeling of a TNFα-specific antibody or antibody derivativecan be accomplished by linking it to an enzyme for use in an enzymeimmunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), whichare methods well known to the person skilled in the art (for exampleCurrent Protocols in Immunology, Coligan et al. Eds, John Wiley & Sons,2005).

By radioactively labeling the TNFα-specific antibodies or antibodyderivatives, it is possible to detect TNF-α through the use ofradioimmune assay (RIA) (see for example, Work et al., LaboratoryTechniques and Biochemistry in Molecular Biology, North HollandPublishing Company, N.Y. (1978). The radioisotope can be detected by theuse of a gamma counter or a scintillation counter or by autoradiography.Particularly useful isotopes are ³H, ¹³¹I, ³⁵S, ¹⁴C, and preferably¹²⁵I.

The antibodies or antibody derivatives of the present invention can alsobe labeled with fluorescent labeling compounds such as fluorescein,isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine, or with chemiluminescent compoundssuch as luminol, isoluminol, theromatic acridinium ester, imidazolacridinium salt and oxalate ester.

Labeling and detection protocols are well known to the person skilled inthe art. For example, they are available from Using Antibodies: ALaboratory Manual: Portable Protocol NO. I (Harlow, E. and Lane, D.,1998).

Labeled antibodies or antibody derivatives of the present invention areuseful for diagnostic purposes, in particular detection of TNFα in abiological sample removed from a patient. Any sample containing TNFα canbe used, e.g. biological fluids like blood, serum, lymph, urine,inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissueextract or homogenate, and the like, or histological specimens for insitu detection.

Pharmaceutical Preparations

Definitions: The term “pharmaceutical formulation” refers topreparations which are in such form as to permit the biological activityof the antibody or antibody derivative to be unequivocally effective,and which contain no additional components which are toxic to thesubjects to which the formulation would be administered.“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed.

A “stable” formulation is one in which the antibody or antibodyderivative therein essentially retains its physical stability and/orchemical stability and/or biological activity upon storage. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. Preferably, the formulation is stable at room temperature (about30° C.) or at 40° C. for at least 1 month and/or stable at about 2-8° C.for at least 1 year for at least 2 years. Furthermore, the formulationis preferably stable following freezing (to, e.g., −70° C.) and thawingof the formulation.

An antibody or antibody derivative “retains its physical stability” in apharmaceutical formulation if it shows no signs of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

An antibody or antibody derivative “retains its chemical stability” in apharmaceutical formulation, if the chemical stability at a given time issuch that the protein is considered to still retain its biologicalactivity as defined below. Chemical stability can be assessed bydetecting and quantifying chemically altered forms of the protein.Chemical alteration may involve size modification (e.g. clipping) whichcan be evaluated using size exclusion chromatography, SDS-PAGE and/ormatrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI/TOF MS), for example. Other types of chemicalalteration include charge alteration (e.g. occurring as a result ofdeamidation) which can be evaluated by ion-exchange chromatography, forexample.

An antibody or antibody derivative “retains its biological activity” ina pharmaceutical formulation, if the biological activity of the antibodyat a given time is within about 10% (within the errors of the assay) ofthe biological activity exhibited at the time the pharmaceuticalformulation was prepared as determined in an antigen binding assay, forexample. Other “biological activity” assays for antibodies areelaborated herein below.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and non-reducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kD (e.g. in the range from about 120 to about 400 kD). A“reducing sugar” is one which contains a hemiacetal group that canreduce metal ions or react covalently with lysine and other amino groupsin proteins and a “non-reducing sugar” is one which does not have theseproperties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Non-reducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof. Where it is desired that the formulation is freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes theantibody in the formulation. Non-reducing sugars such as sucrose andtrehalose are the preferred polyols herein, with trehalose beingpreferred over sucrose, because of the superior solution stability oftrehalose.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention has a pH in the range from about 4.5 to about6.0; preferably from about 4.8 to about 5.5; and most preferably has apH of about 5.0. Examples of buffers that will control the pH in thisrange include acetate (e.g. sodium acetate), succinate (such as sodiumsuccinate), gluconate, histidine, citrate and other organic acidbuffers. Where a freeze-thaw stable formulation is desired, the bufferis preferably not phosphate.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an antibody or antibody derivativerefers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody or antibody derivativeis effective. A “disease/disorder” is any condition that would benefitfrom treatment with the antibody or antibody derivative. This includeschronic and acute disorders or diseases including those pathologicalconditions which predispose the mammal to the disorder in question.

A “preservative” is a compound which can be included in the formulationto essentially reduce bacterial action therein, thus facilitating theproduction of a multi-use formulation, for example. Examples ofpotential preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferredpreservative herein is benzyl alcohol.

The present invention also provides pharmaceutical compositionscomprising one or more antibodies or antibody derivative compounds,together with at least one physiologically acceptable carrier orexcipient. Pharmaceutical compositions may comprise, for example, one ormore of water, buffers (e.g., neutral buffered saline or phosphatebuffered saline), ethanol, mineral oil, vegetable oil,dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, adjuvants, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione and/or preservatives. As noted above, other activeingredients may (but need not) be included in the pharmaceuticalcompositions provided herein.

A carrier is a substance that may be associated with an antibody orantibody derivative prior to administration to a patient, often for thepurpose of controlling stability or bioavailability of the compound.Carriers for use within such formulations are generally biocompatible,and may also be biodegradable. Carriers include, for example, monovalentor multivalent molecules such as serum albumin (e.g., human or bovine),egg albumin, peptides, polylysine and polysaccharides such asaminodextran and polyamidoamines. Carriers also include solid supportmaterials such as beads and microparticles comprising, for example,polylactate polyglycolate, poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose or dextran. A carrier may bear the compounds ina variety of ways, including covalent bonding (either directly or via alinker group), noncovalent interaction or admixture.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal, rectalor parenteral administration. In certain embodiments, compositions in aform suitable for oral use are preferred. Such forms include, forexample, pills, tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs. Within yet other embodiments, compositions providedherein may be formulated as a lyophilizate. The term parenteral as usedherein includes subcutaneous, intradermal, intravascular (e.g.,intravenous), intramuscular, spinal, intracranial, intrathecal andintraperitoneal injection, as well as any similar injection or infusiontechnique.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and may contain one or more agents, such as sweeteningagents, flavoring agents, coloring agent, and preserving agents in orderto provide appealing and palatable preparations. Tablets contain theactive ingredient in admixture with physiologically acceptableexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example, inert diluents (e.g., calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate), granulating and disintegrating agents (e.g., corn starch oralginic acid), binding agents (e.g., starch, gelatin or acacia) andlubricating agents (e.g., magnesium stearate, stearic acid or talc). Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monosterate or glyceryl distearatemay be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent(e.g., calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium (e.g., peanut oil, liquid paraffin or olive oil). Aqueoussuspensions contain the antibody or antibody derivative in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients include suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions may also comprise one or more preservatives, for exampleethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, and one or more sweetening agents, such assucrose or saccharin. Syrups and elixirs may be formulated withsweetening agents, such as glycerol, propylene glycol, sorbitol, orsucrose. Such formulations may also comprise one or more demulcents,preservatives, flavoring agents, and/or coloring agents.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil (e.g., arachis oil, olive oil, sesame oil, or coconutoil) or in a mineral oil such as liquid paraffin. The oily suspensionsmay contain a thickening agent such as beeswax, hard paraffin, or cetylalcohol. Sweetening agents, such as those set forth above, and/orflavoring agents may be added to provide palatable oral preparations.Such suspensions may be preserved by the addition of an anti-oxidantsuch as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil (e.g., olive oil orarachis oil), a mineral oil (e.g., liquid paraffin), or a mixturethereof. Suitable emulsifying agents include naturally-occurring gums(e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides(e.g., soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol), anhydrides (e.g., sorbitan monoleate), andcondensation products of partial esters derived from fatty acids andhexitol with ethylene oxide (e.g., polyoxyethylene sorbitan monoleate).An emulsion may also comprise one or more sweetening and/or flavoringagents.

The pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension in which the modulator, depending onthe vehicle and concentration used, is either suspended or dissolved inthe vehicle. Such a composition may be formulated according to the knownart using suitable dispersing, wetting agents and/or suspending agentssuch as those mentioned above. Among the acceptable vehicles andsolvents that may be employed are water, 1,3-butanediol, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils may be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectible compositions, and adjuvants such aslocal anesthetics, preservatives and/or buffering agents can bedissolved in the vehicle.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of modulator following administration). Such formulations maygenerally be prepared using well known technology and administered by,for example, oral, rectal, or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofmodulator release. The amount of an antibody or antibody derivativecontained within a sustained release formulation depends upon, forexample, the site of implantation, the rate and expected duration ofrelease and the nature of the disease/disorder to be treated orprevented.

Antibody or antibody derivatives provided herein are generallyadministered in an amount that achieves a concentration in a body fluid(e.g., blood, plasma, serum, CSF, synovial fluid, lymph, cellularinterstitial fluid, tears or urine) that is sufficient to detectablybind to TNFα and prevent or inhibit TNFα associated diseases/disorders.A dose is considered to be effective if it results in a discerniblepatient benefit as described herein. Preferred systemic doses range fromabout 0.1 mg to about 140 mg per kilogram of body weight per day (about0.5 mg to about 7 g per patient per day), with oral doses generallybeing about 5-20 fold higher than intravenous doses. The amount ofantibody or antibody derivative that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Dosage unitforms will generally contain between from about 1 mg to about 500 mg ofan active ingredient.

Pharmaceutical compositions may be packaged for treating conditionsresponsive to an antibody or antibody derivative directed to TNF-α.Packaged pharmaceutical compositions may include a container holding aeffective amount of at least one antibody or antibody derivative asdescribed herein and instructions (e.g., labeling) indicating that thecontained composition is to be used for treating a disease/disorderresponsive to one antibody or antibody derivative followingadministration in the patient.

The antibodies or antibody derivatives of the present invention can alsobe chemically modified. Preferred modifying groups are polymers, forexample an optionally substituted straight or branched chain polyalkene,polyalkenylene, or polyoxyalkylene polymer or a branched or unbranchedpolysaccharide. Such effector group may increase the half-live of theantibody in vivo. Particular examples of synthetic polymers includeoptionally substituted straight or branched chain poly(ethyleneglycol)(PEG), poly(propyleneglycol), poly(vinylalcohol) or derivatives thereof.Particular naturally occurring polymers include lactose, amylose,dextran, glycogen or derivatives thereof. The size of the polymer may bevaried as desired, but will generally be in an average molecular weightrange from 500 Da to 50000 Da. For local application where the antibodyis designed to penetrate tissue, a preferred molecular weight of thepolymer is around 5000 Da. The polymer molecule can be attached to theantibody, in particular to the C-terminal end of the Fab fragment heavychain via a covalently linked hinge peptide as described in WO0194585.Regarding the attachment of PEG moieties, reference is made to“Poly(ethyleneglycol) Chemistry, Biotechnological and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York and“Bioconjugation Protein Coupling Techniques for the BiomedicalSciences”, 1998, M. Aslam and A. Dent, Grove Publishers, New York.

Preparation of the Formulation

After preparation of the antibody or antibody derivative of interest asdescribed above, the pharmaceutical formulation comprising it isprepared. The antibody to be formulated has not been subjected to priorlyophilization and the formulation of interest herein is an aqueousformulation. Preferably the antibody or antibody derivative in theformulation is an antibody fragment, such as an scFv. Thetherapeutically effective amount of antibody present in the formulationis determined by taking into account the desired dose volumes andmode(s) of administration, for example. From about 0.1 mg/ml to about 50mg/ml, preferably from about 0.5 mg/ml to about 25 mg/ml and mostpreferably from about 2 mg/ml to about 10 mg/ml is an exemplary antibodyconcentration in the formulation.

An aqueous formulation is prepared comprising the antibody or antibodyderivative in a pH-buffered solution The buffer of this invention has apH in the range from about 4.5 to about 6.0, preferably from about 4.8to about 5.5, and most preferably has a pH of about 5.0. Examples ofbuffers that will control the pH within this range include acetate (e.g.sodium acetate), succinate (such as sodium succinate), gluconate,histidine, citrate and other organic acid buffers.

The buffer concentration can be from about 1 mM to about 50 mM,preferably from about 5 mM to about 30 mM, depending, for example, onthe buffer and the desired isotonicity of the formulation. The preferredbuffer is sodium acetate (about 10 mM), pH 5.0.

A polyol, which acts as a tonicifier and may stabilize the antibody, isincluded in the formulation. In preferred embodiments, the formulationdoes not contain a tonicifying amount of a salt such as sodium chloride,as this may cause the antibody or antibody derivative to precipitateand/or may result in oxidation at low pH. In preferred embodiments, thepolyol is a non-reducing sugar, such as sucrose or trehalose. The polyolis added to the formulation in an amount which may vary with respect tothe desired isotonicity of the formulation. Preferably the aqueousformulation is isotonic, in which case suitable concentrations of thepolyol in the formulation are in the range from about 1% to about 15%w/v, preferably in the range from about 2% to about 10% whv, forexample. However, hypertonic or hypotonic formulations may also besuitable. The amount of polyol added may also alter with respect to themolecular weight of the polyol. For example, a lower amount of amonosaccharide (e.g. mannitol) may be added, compared to a disaccharide(such as trehalose).

A surfactant is also added to the antibody or antibody derivativeformulation. Exemplary surfactants include nonionic surfactants such aspolysorbates (e.g. polysorbates 20, 80 etc) or poloxamers (e.g.poloxamer 188). The amount of surfactant added is such that it reducesaggregation of the formulated antibody/antibody derivative and/orminimizes the formation of particulates in the formulation and/orreduces adsorption. For example, the surfactant may be present in theformulation in an amount from about 0.001% to about 0.5%, preferablyfrom about 0.005% to about 0.2% and most preferably from about 0.01% toabout 0.1%.

In one embodiment, the formulation contains the above-identified agents(i.e. antibody or antibody derivative, buffer, polyol and surfactant)and is essentially free of one or more preservatives, such as benzylalcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In anotherembodiment, a preservative may be included in the formulation,particularly where the formulation is a multidose formulation. Theconcentration of preservative may be in the range from about 0.1% toabout 2%, most preferably from about 0.5% to about 1%. One or more otherpharmaceutically acceptable carriers, excipients or stabilizers such asthose described in Remington's Pharmaceutical Sciences 21st edition,Osol, A. Ed. (2006) may be included in the formulation provided thatthey do not adversely affect the desired characteristics of theformulation. Acceptable carriers, excipients or stabilizers arenon-toxic to recipients at the dosages and concentrations employed andinclude; additional buffering agents; co-solvents; antioxidantsincluding ascorbic acid and methionine; chelating agents such as EDTA;metal complexes (e.g. Zn-protein complexes); biodegradable polymers suchas polyesters; and/or salt-forming counterions such as sodium.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation.

Administration of the Formulation

The formulation is administered to a mammal in need of treatment withthe antibody, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. In preferred embodiments, the formulationis administered to the mammal by intravenous administration. For suchpurposes, the formulation may be injected using a syringe or via an IVline, for example.

The appropriate dosage (“therapeutically effective amount”) of theantibody will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, the type ofantibody used, and the discretion of the attending physician. Theantibody or antibody derivative is suitably administered to the patentat one time or over a series of treatments and may be administered tothe patent at any time from diagnosis onwards. The antibody or antibodyderivative may be administered as the sole treatment or in conjunctionwith other drugs or therapies useful in treating the condition inquestion.

As a general proposition, the therapeutically effective amount of theantibody or antibody derivative administered will be in the range ofabout 0.1 to about 50 mg/kg of patent body weight whether by one or moreadministrations, with the typical range of antibody used being about 0.3to about 20 mg/kg, more preferably about 0.3 to about 15 mg/kg,administered daily, for example. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the aqueous pharmaceuticalformulation of the present invention and optionally providesinstructions for its use. Suitable containers include, for example,bottles, vials and syringes. The container may be formed from a varietyof materials such as glass or plastic. An exemplary container is a 3-20cc single use glass vial. Alternatively, for a multidose formulation,the container may be 3-100 cc glass vial. The container holds theformulation and the label on, or associated with, the container mayindicate directions for use. The article of manufacture may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

Generating the Antibodies of the Present Invention

The antibodies or antibody derivatives of the present invention may begenerated using routine techniques in the field of recombinant genetics.Knowing the sequences of the polypeptides, the cDNAs encoding them canbe generated by gene synthesis. These cDNAs can be cloned into suitablevector plasmids. Once the DNA encoding a VL and/or a VH domain areobtained, site directed mutagenesis, for example by PCR using mutagenicprimers, can be performed to obtain various derivatives. The best“starting” sequence can be chosen depending on the number of alterationsdesired in the VL and/or VH sequences. A preferred sequence is the TB-Asequences and its derivatives, e.g. scFv sequences or Fab fusion peptidesequences, may be chosen as templates for PCR driven mutagenesis and/orcloning.

Standard cloning and mutagenesis techniques well known to the personskilled in the art can be used to attach linkers, shuffle domains orconstruct fusions for the production of Fab fragments. Basic protocolsdisclosing the general methods of this invention are described inMolecular Cloning, A Laboratory Manual (Sambrook & Russell, 3^(rd) ed.2001) and in Current Protocols in Molecular Biology (Ausubel et al.,1999).

The DNA sequence harboring a gene encoding a scFv polypeptide, or in thecase of Fab fragments, encoding either two separate genes or abi-cistronic operon comprising the two genes for the VL-Cκ. and theVH-CH1 fusions (FIG. 2) are cloned in a suitable expression vector,preferably one with an inducible promoter. Care must be taken that infront of each gene an appropriate ribosome binding site (RBS in FIG. 2)is present that ensures translation. It is to be understood that theantibodies of the present invention comprise the disclosed sequencesrather than they consist of them. For example, cloning strategies mayrequire that a construct is made from which an antibody with one or afew additional residues at the N-terminal end are present. Specifically,the methionine derived from the start codon may be present in the finalprotein in cases where it has not been cleaved posttranslationally. Mostof the constructs for scFv antibodies give rise to an additional alanineat the N-terminal end. In a preferred embodiment of the presentinvention, an expression vector for periplasmic expression in E. coli ischosen (Krebber, 1997). Said vector comprises a promoter in front of acleavable signal sequence. The coding sequence for the antibody peptideis then fused in frame to the cleavable signal sequence. This allows thetargeting of the expressed polypeptide to the bacterial periplasm wherethe signal sequence is cleaved. The antibody is then folded. In the caseof the Fab fragments, both the VL-κ. and the VH-CH1 fusions peptidesmust be linked to an export signal. The covalent S—S bond is formed atthe C-terminal cysteines after the peptides have reached the periplasm.If cytoplasmic expression of antibodies is preferred, said antibodiesusually can be obtained at high yields from inclusion bodies, which canbe easily separated from other cellular fragments and protein. In thiscase the inclusion bodies are solubilized in a denaturing agent such ase.g. guaridine hydrochloride (GndHCl) and then refolded by renaturationprocedures well known to those skilled in the art. Plasmids expressingthe scFv or Fab polypeptides are introduced into a suitable host,preferably a bacterial, yeast or mammalian cell, most preferably asuitable E. coli strain as for example JM83 for periplasmic expressionor BL21 for expression in inclusion bodies. The polypeptide can beharvested either from the periplasm or form inclusion bodies andpurified using standard techniques such as ion exchange chromatography,reversed phase chromatography, affinity chromatography and/or gelfiltration known to the person skilled in the art.

The antibodies or antibody derivatives of the present invention can becharacterized with respect to yield, solubility and stability in vitro.Binding capacities towards TNFα, preferably towards human TNFα, can betested in vitro by ELISA or surface plasmon resonance (BIACore), usingrecombinant human TNFα as described in WO9729131, the latter method alsoallowing to determine the k_(off) rate constant, which should preferablybe less than 10⁻³ s⁻¹. K_(d) values of ≦10 nM are preferred.

In vivo neutralizing activity of an antibody or antibody derivative ofthe present invention can be estimated using the L929 cytotoxicityassay. Human recombinant TNFα exerts a cytotoxic effect towards culturedmouse L929 fibroblast cells in a concentration-dependent manner. ThisTNFα-induced cytotoxicity can be inhibited by TNFα neutralizingantibodies (Miring, 1994). A preferred IC₅₀ value corresponding to ahalf-maximal inhibitor concentration is ≦100 ng ml⁻¹.

As TNFα has a proven pathophysiological role in various human diseases,in particular inflammatory disorders, immune and immune-regulateddisorders, infections causing septic, endotoxic and cardiovascularshock, neurodegenerative diseases, and malignant diseases. As TNFα issuspected to play a disease-relevant role in a steadily growing numberof additional human diseases, it is difficult to give a comprehensivelist of indications that also ensures a complete representation of thespectrum of clinical applications for TNFα inhibitors in the future.Therefore, the antibodies or antibody derivatives of the presentinvention can be applied to treat the diseases listed in the followingcatalogue, which is not to be considered as a complete or exclusivelist. Other diseases not mentioned specifically, which directly orindirectly are influenced by TNFα, are also included.

Autoimmune or Chronic Inflammation:

Chronic and/or autoimmune states of inflammation in general, immunemediated inflammatory disorders in general, inflammatory CNS disease,inflammatory diseases affecting the eye, joint, skin, mucuous membranes,central nervous system, gastrointestinal tract, urinary tract or lung,states of uveitis in general, retinitis, HLA-B27+ uveitis, Behcet'sdisease, dry eye syndrome, glaucoma, Sjögren syndrome, diabetes mellitus(incl. diabetic neuropathy), insulin resistance, states of arthritis ingeneral, rheumatoid arthritis, osteoarthritis, reactive arthritis andReiter's syndrome, juvenile arthritis, ankylosing spondylitis, multiplesclerosis, Guillain-Barre syndrome, myasthenia gravis, amyotrophiclateral sclerosis, sarcoidosis, glomerulonephritis, chronic kidneydisease, cystitis, Psoriasis (incl. psoriatic arthritis), hidradenitissuppurativa, panniculitis, pyoderma gangrenosum, SAPHO syndrome(synovitis, acne, pustulosis, hyperostosis and osteitis), acne, Sweet'ssydrome, pemphigus, Crohn's disease (incl. extraintestinalmanifestastations), ulcerative colitis, asthma bronchiale,hypersensitivity pneumonitis, general allergies, allergic rhinitis,allergic sinusitis, chronic obstructive pulmonary disease (COPD), lungfibrosis, Wegener's granulomatosis, Kawasaki syndrome, Giant cellarteritis, Churg-Strauss vasculitis, polyarteritis nodosa, burns, graftversus host disease, host versus graft reactions, rejection episodesfollowing organ or bone marrow transplantation, sytemic and local statesof vasculitis in general, systemic and discoid lupus erythematodes,polymyositis and dermatomyositis, sclerodermia, pre-eclampsia, acute andchronic pancreatitis, viral hepatitis, alcoholic hepatitis.

Acute Inflammation and/or Prevention of Postsurgical or PosttraumaticInflammation and Pain:

Prevention of postsurgical inflammation in general, eye surgery (e.g.cataract (eye lens replacement) or glaucoma surgery), joint surgery(incl. arthroscopic surgery), surgery at joint-related structures (e.g.ligaments), oral and/or dental surgery, minimally invasivecardiovascular procedures (e.g. PTCA, atherectomy, stent placement),laparoscopic and/or endoscopic intra-abdominal and gynecologicalprocedures, endoscopic urological procedures (e.g. prostate surgery,ureteroscopy, cystoscopy, interstitial cystitis), perioperativeinflammation (prevention) in general.

Neurological and Neurodegenarative Diseases:

Alzheimer disease, Parkinson's disease, Huntington's disease, Bell′palsy, Creutzfeld-Jakob disease.

Cancer:

Cancer-related osteolysis, cancer-related inflammation, cancer-relatedpain, cancer-related cachexia, bone metastases.

Pain:

Acute and chronic forms of pain, irrespective whether these are causedby central or peripheral effects of TNFα and whether they are classifiedas inflammatory, nociceptive or neuropathic forms of pain, sciatica, lowback pain, carpal tunnel syndrome, complex regional pain syndrome(CRPS), gout, postherpetic neuralgia, fibromyalgia, local pain states,chronic pain syndroms due to metastatic tumor, dismenorrhea.

Infection:

Bacterial, viral or fungal sepsis, tuberculosis, AIDS.

Cardiovascular Disease:

Atherosclerosis, coronary artery disease, hypertension, dyslipidemia,heart insufficiency and chronic heart failure.

In a preferred embodiment of the present invention, treatment ofosteoarthritis or uveitis or inflammatory bowel disease can be achievedwith the antibodies or antibody derivatives of the present invention.

The present invention also provides a pharmaceutical compositioncomprising an antibody or antibody derivative molecule of the presentinvention in combination with a pharmaceutically acceptable excipient,diluent or carrier.

The pharmaceutical composition should preferably comprise atherapeutically effective amount of the antibody of the presentinvention, i.e. an amount of said antibody that is needed to treat,ameliorate or prevent the TNFα-related disease or condition, or toexhibit a detectable therapeutic or preventive effect. Thetherapeutically effective dose can be estimated either in cell cultureassays or in animal models, usually in rodents, rabbits, dogs, pigs orprimates. The animal model may also be used to determine the appropriateconcentration range and route of administration. A suitable animal modelto observe an effect of the antibody or antibody derivative of thepresent invention is a rat model for acute monoarthritis (Bolon et al.(2004), Vet. Pathol. 41:235-243. Human TNFα is injected intraarticularlyinto the knee joint of a rat, leading to an acute, self-limitingmonoarthritis in the injected joint. Bioactivity of an anti-TNFαantibody (derivative) can be quantified by reduction of TNFα-inducedknee joint swelling and or reduction of histological parameters ofinflammation.

As the antibodies or antibody derivatives of the present invention arehighly soluble, high antibody concentrations (60 mg ml⁻¹ or more) enablethe use of small application volumes.

The antibody or antibody derivative of the present invention may beutilised in any therapy where it is desired to reduce the level ofbiological active TNFα present in the human or animal body. The TNFα maybe circulating in the body or be present at an undesirably high levellocalised at a particular site in the body. The present inventionprovides modes for systemic as well as local applications in general,which include, but are not limited to the following ones: peroralapplication, intravenous, subcutaneous, intramuscular, intraarticular,intravitreal, intradermal, or intraparenchymal injection, aerosolinhalation, topical application to the skin, to mucous membranes or toeye, systemic or local release via implantable minipump or local releasevia implantable formulation/device allowing for retarded release,topical application to serosal surfaces, intrathecal or intraventricularapplication, oral application in formulations allowing for controlledintralumenal release in selected parts of the gastrointestinal tract,localized intravasal release from adequate formulation/devices (e.g.stents), local delivery to urinary cyst, localized intralumenal release(e.g. to biliary tract, ureter), or local delivery through endoscopicdevices, or release from contact lenses (contacts). A preferredapplication is a local one such as intraarticular injection or topicapplication e.g. into the eye. For both preferred applications, theantibody of the present invention needs to be in solution.

The present invention also reveals the use of the antibody or antibodyderivative of the present invention for the production of a medicamentfor the treatment of TNFα associated diseases. In this case, theantibody or antibody derivative is comprised in a therapeuticcomposition. Said composition is used as a medicament, most preferablyfor the prevention or therapy of TNFα related diseases.

In another aspect the scFv antibodies of the present invention are usedin gene therapy, in particular in adoptive cellular gene therapy.Autoimmune disorders represent inappropriate immune responses directedat self-tissue. Antigen-specific CD4+ T cells and antigen-presentingdendritic cells (DCs) are important mediators in the pathogenesis ofauto-immune disease and thus are ideal candidates for adoptive cellulargene therapy, an ex vivo approach to therapeutic gene transfer. Usingretrovirally transduced cells and luciferase bioluminescence, Tarner etal. (2003, Ann. N. Y. Acad. Sci. 998:512-519) have demonstrated thatprimary T cells, T cell hybridomas, and DCs rapidly and preferentiallyhome to the sites of inflammation in animal models of multiplesclerosis, arthritis, and diabetes. These cells, transduced withretroviral vectors that drive expression of various “regulatoryproteins” such as interleukins and anti-TNF scFv, deliver theseimmunoregulatory proteins to the inflamed lesions, providing therapy forexperimental autoimmune encephalitis, collagen-induced arthritis, andnonobese diabetic mice. The stable and soluble frameworks of theantibodies or antibody derivatives of the present inventions areparticularly suitable for intracellular delivery of the antigen, forexample when the antibody or antibody derivative is expressed from atransgene carried by a suitable retroviral vector. Adoptive cellulargene therapy leads to localized expression and secretion of theanti-TNFα scFv. Smith et al. (2003) have demonstrated that scFvs derivedfrom a TNFα neutralizing monoclonal antibody (i) can neutralize TNFα invitro and (ii) change the cytokine expression pattern in mice sufferingfrom collagen-induced arthritis locally in the joints, but notsystemically. Alternatively, direct systemic or local injection ofsuitable vectors (e.g. viruses) allowing for continuous expression of ananti-TNFα scFv antibody, is considered as another possible gene therapyapproach.

The sequences of the present invention are the following ones:

SEQ ID NO: 1 VL of TB-ADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQ GTKLEVKRSEQ ID NO: 2 VH of TB-AQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARER GDAMDYWGQGTLVTVSSSEQ ID NO: 3 VL of TB-BDIVLTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKRLIYSAFNRYTGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQDYNSPRTFGQ GTKLEVKRSEQ ID NO: 4 VH of TB-BQVQLVQSGAEVKKPGASVKVSCTASGYSFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARER GDAMDYWGQGTLVTVSSSEQ ID NO: 5 VL of FW2.3DIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYAGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ GTKLEVKRSEQ ID NO: 6 VH of FW2.3QVQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRINPDSGDTIYAQKFQDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARVPRGTYLDPWDYFDYWGQGTLVTVSS SEQ ID NO: 7 VL of TB_L2DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYAGSILQSGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQ GTKLEVKRSEQ ID NO: 8 VL of TB_L3DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ GTKLEVKRSEQ ID NO: 9 VH of TB_H2QVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGRINPDSGDTIYAQKFQDRFTFSLETSASTVYMELTSLTSDDTAVYYCARER GDAMDYWGQGTLVTVSSSEQ ID NO: 10 Linker GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 11 VL of TB-B R46LDIVLTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQDYNSPRTFGQ GTKLEVKRSEQ ID NO: 12 TB-AB DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYSFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKDRVTLTRDTSIGTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS SEQ ID NO: 13 TB-BADIVLTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKRLIYSAFNRYTGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVINSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS SEQ ID NO: 14 Cκ FabTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECSEQ ID NO: 15 CH1 of FabASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFSEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCTSSEQ ID NO: 16 VL of TB-wtDIVMTQTPKFLLVSAGDRVTITCTASQSVSNDVVWYQQKPGQSPKMLMYSAFNRYTGVPDRFTGRGYGTDFTFTISSVQAEDLAVYFCQQDYNSPRTFGG GTKLEIKRSEQ ID NO: 17 VH of TB-wtQIQLVQSGPELKKPGETVKISCKASGYTFTHYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKEHFAFSLETSASTVFLQINNLKNEDTATYFCARER GDAMDYWGQGTSVTVSSSEQ ID NO: 18 TB-A H_K43Q, also named TB-A H43DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 19 TB-A H_F68V, also named TB-A H68DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRVTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 20 TB-A H_F70L/L72R, also named TB-A H70/72DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTLSRETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 21, TB-A H_A76I/S77G, also named TB-A H76/77DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSIGTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 22 TB-A L_L46R, also named TB-A L46DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKRLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 23 TB-A L_R65S, also named TB-A L65DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGSGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVINSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 24 TB-A L_Y67S, also named TB-A L67DIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGSGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 25 VH of TB-A with D66GQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKGRFTFSLETSASTVYMELTSLTSDDTAVYYCARER GDAMDYWGQGTLVTVSSSEQ ID NO: 26 VL of TB-A with V83FDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDFAVYYCQQDYNSPRTFGQ GTKLEVKRSEQ ID NO: 27 VL of TB-A with V83ADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDAAVYYCQQDYNSPRTFGQ GTKLEVKRSEQ ID NO: 28 VH of TB-A H43/70/71/73/77QVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKDRFTLTLDTSAGTVYMELTSLTSDDTAVYYCARER GDAMDYWGQGTLVTVSSSEQ ID NO: 29 VH of TB-A H43/70/71QVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKDRFTLTLETSASTVYMELTSLTSDDTAVYYCARER GDAMDYWGQGTLVTVSSSEQ ID NO: 30 VH of TB-A H11/16/43/66/70/71/73/77/ 93/112QVQLVQSGAEDKKPGGSVKVSCTASGYTFTHYGMNWVRQAPGQGLEWMGWINTYTGEPTYADKFKGRFTLTLDTSAGTVYMELTSLTSDDTATYYCARER GDAMDYWGQGTSVTVSSSEQ ID NO: 31 TB-A H_M48L/F68IDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWLGWINTYTGEPTYADKFKDRITFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 32 TB-A L_V83E H_V79ADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDEAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTAYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 33 TB-A Linker_G2R H_F68LDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGRGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRLTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 34 TB-A H_K43R/F68IDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVINSCTASGYTFTHYGMNWVRQAPGRGLEWMGWINTYTGEPTYADKFKDRITFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS SEQ ID NO: 35 TB-A H_F68LDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRLTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS SEQ ID NO: 36 TB-A H_F68ADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRATFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSSSEQ ID NO: 37 TB-A H_F68V/F70LDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRVTLSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS SEQ ID NO: 38 TB-A H_F70LDIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTLSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS SEQ ID NO: 39 Linker G2RGRGGSGGGGSGGGGSGGGGS SEQ ID NO: 40 TB-ADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQQRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTISSLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

Experiment 1 Construction of scFv Antibodies

The starting material for the generation of humanised anti-human TNFαantibodies or antibody derivatives, such as single-chain fragments(scFv) or Fab fragments, was the murine monoclonal antibody Di62. Thesequences of the variable region of the light chain and the heavy chainare disclosed in Döring et al. (1994, Mol. Immunol. 31:1059-1067). Theproperties of this monoclonal antibody are also discussed in the samepublication. Briefly, Di62 specifically binds to human TNFα in aconcentration-dependent manner. It is a high affinity antibody (Kd=0.4nM) and can block TNFα binding to its receptor. In addition, Di62inhibits human TNFα-induced cytotoxicity in mouse L929 cells.

Based on its published sequence Di62 was constructed in the form of asingle-chain antibody derivative (scFv) in the orientation VL-linker-VH,in which the linker sequence is composed of four repeats of four glycineand one serine residue (Gly₄Ser)₄ (SEQ ID NO: 10). Herein, this scFv isreferred to as TB-wt, with a VL of SEQ ID NO:16 and a VH of SEQ IDNO:17.

To humanise this antibody derivative for the purpose of a) render itmore similar to human sequences in order to minimise potentialimmunogenicity, and b) render it more stable and soluble, TB-wt CDRsequences were grafted on stable and soluble human frameworks (Auf derMaur et al. (2001), FEBS Lett. 508:407-412; Auf der Maur et al. (2004),Methods 34:215-224). The human VL-kappa subgroup I and VH subgroup Iwere identified as nearest human subfamily. The appropriate acceptorframework was chosen from a pool of human VL and VH sequences, selectedfor advantageous biochemical and biophysical properties, such as forexample stability, solubility, and expression properties (Auf der Maur,et al. (2001), FEBS Lett. 508:407-412; Auf der Maur, et al. (2004),Methods 34:215-224). The isolation and properties of these antibodyframeworks are described in WO03097697/EP1506236. From this pool, asingle-chain antibody framework with undefined antigen bindingproperties was identified as suitable acceptor. This acceptor consistsof a human VL-kappa I domain (SEQ ID NO:5) in combination with a humanVHI domain (SEQ ID NO:6). Herein, this acceptor framework is referred toas FW2.3. Among 81 VL framework residues, TB-wt and FW2.3 share 55identical amino acid residues, which amounts to 67% identity. Among the87 VH framework residues, TB-wt and FW2.3 have 55 identical residues,corresponding to 63% identity. Both single-chain antibody derivativeshave identical CDR lengths, apart from the VH-CDR3, which is longer inFW2.3. The amino acid composition within the CDR residues is differentfor both scFvs. Various methods for the humanisation of antibodyvariable domains are described (Riechmann et al. (1998), Nature332:323-327; Padlan, E. A. (1991). Mol. Immunol. 28:489-498; Roguska etal. (1994), Proc. Natl. Acad. Sci. USA 91:969-973; Gonzales et al.(2005), Tumor Biol. 26:31-43; Ewert, S., et al. (2004), Methods34:184-199). The minimal approach, namely the conservative transfer ofall mouse CDR loops from TB-wt onto FW2.3 was carried out first. Theresulting scFv is referred to as TB-B and has the VL sequence of SEQ IDNO:3 and the VH sequence of SEQ ID NO:4. The CDR-loops in TB-wt weredefined according to the Kabat numbering scheme (Kabat et al. (1991),Sequences of Proteins of Immunological Interest, 5^(th) Ed, Natl. Inst.Health, Bethesda, Md.) and confine the following residues (see FIG. 1):

-   VL-   CDR1: L24-L34 (same Kabat numbering)-   CDR2: L50-L56 (same Kabat numbering)-   CDR3: L89-L97 (same Kabat numbering)-   VH-   CDR1: H31-H35 (same Kabat numbering)-   CDR2: H50-H66 (Kabat numbering H50-H65)-   CDR3: H99-H106 (Kabat numbering H95-H102).

This antibody was unable to bind TNFα efficiently (FIG. 4A). The nextstep was to determine which residue or residues from these componentsshould be substituted to optimise the properties of the resultinghumanised antibody.

Since substitution of human amino acid residues with other amino acidsshould be minimised because introduction of foreign amino acid sequencesincreases the risk of immunogenicity of the antibody or antibodyderivative in humans (Gonzales et al. (2005), Tumor Biol. 26:31-43),several variants were constructed. One of said variants—herein referredto as TB-B L46 (VL SEQ ID NO:11; VH SEQ ID NO:4) was constructed withthe aim to minimize the risk of being immunogenic but still showingsufficient binding activity. This variant is based on TB-B and containsone single amino acid change at position 46 in VL, namely R→L. Thisamino acid is located within the upper core of the light chain and takespart in the dimer interface. It is involved in defining the conformationof L-CDR1 and has an influence on VH/VL packing. It was reported that aleucine residue is favoured at this particular position(PCT/US03/19333). In contrast to TB-B, the scFv TB-B L46 retains someTNFα-specific binding (FIG. 4A; K_(d)≈100 nM)).

In order to further improve the TNFα-binding activity, one or morefurther exchanges were made at one or more of the VL residues 4, 46, 65,67, 70, and/or VH residues 28, 43, 68, 70, 71, 72, 73, 76, 77. Herein,the variant with exchanges in all positions is referred to as TB-A (VLSEQ ID NO:1; VH SEQ ID NO:2).

Furthermore, regarding particular anti-TNFα antibodies of the presentinvention, competition assays with peptides derived from L-CDR1 andL-CDR2 showed that both CDR loops are important for the binding of thescFv to the antigen (Döring et al. (1994), Mol. Immunol. 31:1059-1067).

In further experiments aimed at minimising the number of non-humanresidues required to retain binding and at optimising biophysicalproperties (stability and solubility), systematic mutagenesis and domainshuffling was applied allowing to elucidate the functional differencesbetween mouse and humanised VL and VH domains.

Two variants were obtained by domain shuffling. The first variant iscomposed of the VL domain from TB-A connected via a glycine serinelinker (SEQ ID NO:10) with the VH domain from TB-B, resulting in TB-AB(SEQ ID NO:12). The second variant, TB-BA, is the reverse of the firstvariant, namely, the VL domain from TB-B in combination with the VHdomain of TB-A (SEQ ID NO:13).

Additional variants were generated by systematic mutagenesis of TB-A.FIG. 1 shows a sequence comparison of the VL and VH sequences of TB-Aand TB-B. A total of 14 framework residues differ between TB-A and TB-B(asterisks). Only five of them, VL residues 4 and 70, and VH residues28, 71, and 73 show only minor differences in size and property andtherefore were not considered for mutagenesis at this point. Thefollowing framework positions were replaced with the corresponding aminoacid from TB-B. These single or double mutants of TB-A are as follows:

TB-A H43 K→Q interface (SEQ ID NO: 18) TB-A H68 F→V outer loop VH (SEQID NO: 19) TB-A H70/72 F→L,L→R outer loop VH (SEQ ID NO: 20) TB-A H76/77A→I,S→G outer loop VH (SEQ ID NO: 21) TB-A L46 L→R interface (SEQ ID NO:22) TB-A L65 R→S outer loop VL (SEQ ID NO: 23) TB-A L67 Y→S outer loopVL (SEQ ID NO: 24)

Many factors can influence the immunogenicity of an antibody or antibodyderivative (Gonzales et al. (2005), Tumor Biol. 26:31-43). To furtherreduce the non-human content of the variable regions of the humanisedTB-A scFv, the murine CDR2 and CDR3 loops of VL and murine CDR2 loop ofVH were exchanged with the corresponding human CDR loops from FW2.3. Theresulting constructs herein are referred to as TB_L2 (SEQ ID NO:7),TB_L3 (SEQ ID NO:8), and TB_H2 (SEQ ID NO:9), respectively.

The cDNAs encoding the murine single-chain version of the monoclonalantibody Di62, and the two humanised versions TB-B and TB-A weregenerated by gene synthesis. All the point mutations in the othervariants (TB-B L46, TB-A H43, TB-A H67, TB-A H69/71, TB-A H75/76, TB-AL46, TB-A L65, TB-A L67, TB-A V83F, TB-A V83A, TB-A D66G) wereintroduced by PCR-driven site-directed mutagenesis following standardcloning procedures. Exchange of the murine CDR loops of TB-A with thehuman CDR loops from FW2.3 was accomplished using PCR and state of theart cloning procedures. The cDNA encoding all the VH TB-A variations asdisclosed in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 were realizedby complete gene synthesis.

Some further preferred TB-A are disclosed in SEQ ID NO: 31 to SEQ ID NO:38. These antibodies were found to be particularly stable and soluble,as shown below in Table I.

All scFv fragments were cloned into an expression vector for periplasmicproduction in E. coli (Krebber et al. (1997), J. Immunol. Methods201:35-55).

In addition to the single-chain antibody derivatives (scFvs) describedabove, the corresponding Fab fragments were generated as follows.Selected light chain variable domains (VL) were fused to the constantregion of a human Ig kappa chain, while the suitable heavy chainvariable domains (VH) were fused to the first (N-terminal) constantdomain (CH1) of a human IgG. Both human constant domains were amplifiedby PCR from a human spleen cDNA library and resulted in the sequence SEQID NO:14 for cκ- and SEQ ID NO:15 for CH1.

Experiment 2 Expression, Production and Stability of Humanised scFv orFab Antibodies

Plasmids encoding TB-wt, its humanised derivatives, or Fab fragmentswere introduced into a suitable E. coli strain (e.g. JM83) forperiplasmic expression. The scFv variants were also expressed asinclusion bodies, for example in the BL21 E. coli strain. Functionalsingle-chain antibodies were obtained by refolding of inclusion bodiesand subsequent purification by, for example, gel filtration.

The expression yields upon periplasmic expression of the scFvs rangedbetween 0.5 mg up to 12 mg per litre culture under standard laboratorycultivation conditions (dYT medium, with approx. 3 hours induction timeat 30° C., shaking at 200 rpm) with conventional shaking flasks. Ingeneral, we observed that, as expected from our previous analysis offrameworks selected for stability and solubility (Auf der Maur, et al.(2004), Methods 34:215-224), the closer the sequence of a humanisedderivative is to the sequence of the acceptor framework (FW2.3), thehigher is the yield obtained upon expression in bacteria. For example,the yield obtained from expression of TB-B is far better than thatobtained from expression of TB-A. In accordance with these findings,reducing the number of differing amino acid residues present in TB-A hada positive effect toward the expression yields (FIG. 3A).

Another important characteristic of the antibodies or antibodyderivatives of the present invention is their solubility. FIG. 3B showsthe superiority of the framework TB-A over the donor framework (TB-wt)in terms of solubility in phosphate buffered saline. In analytical gelfiltration, TB-A migrates predominantly in a monomer state (peak at 70ml) whereas TB-wt shows a strong tendency to form aggregates (peak at 50ml). In addition to that, maximal solubility of TB-A and certainderivatives thereof was assessed by PEG precipitation (Athat DH. et al.JBC. 1981, 256; 23. 12108-12117). Briefly, the apparent solubility oftest proteins was measured in the presence of polyethylene glycol 3000(PEG3000). Solubility curves were determined by measuring proteinconcentration in the supernatant of centrifuged protein-PEG300 mixtures.All curves showed a linear dependence of log S (mg/ml) on PEG3000concentration (%, w/v). Maximal solubility (S_(max)) of a test proteinwas estimated by extrapolation of the linear regression towards 0% PEG(Tab. I). For TB-A was S. calculated to be about 70 mg/ml. All testproteins showed exceptionally good solubility. In a second approach amethod called self-interaction chromatography (SIC) was applied toassess intermolecular attraction/repulsion of TB-A (SEQ ID NO:40), TB-ALinker_G2R H_F68L (SEQ ID NO:33), TB-A H_K43R/F68I (SEQ ID NO:34) andTB-A H_F68L (SEQ ID NO:35) at a concentration of 1 mg/ml in PBS (50 mMphosphate pH 6.5, 150 mM NaCl). In this method the protein of interestis immobilized onto a porous stationary phase and packed into a column.Interactions between the free (mobile phase) and immobilized protein aredetected as shifts in the retention volume. The protein osmotic secondvirion coefficient B₂₂, which is a measure for intermolecularattraction/repulsion, was calculated according to Tessier, P M et al.Biophys. J. 2002, 82: 1620-1632 (Tab. I). The more positive B₂₂, thelower is the intermolecular attraction of the test protein and,therefore, the higher is its solubility. Due to the high similarity oftest protein sequences it was assumed that B₂₂ values of the differentproteins can be directly compared to each other.

TABLE I Solubility features of TB-A derivatives Sequence pI Log S_(max)B₂₂ value (SIC) TB-A 7.8 1.84 ± 0.13 −24.5 × 10⁻⁴ ± 3.8 × 10⁻⁴ TB-AH_M48L/F68I 7.8 nd nd TB-A L_V83E H_V79A 6.58 nd nd TB-A Linker_G2R 8.21.91 ± 0.09   1.59 × 10⁻³ ± 5.9 × 10⁻⁵ H_F68L TB-A H_K43R/F68I 7.8 1.86± 0.02   1.28 × 10⁻³ ± 3.0 × 10⁻⁴ TB-A H_F68L 7.8 1.88 ± 0.07   1.06 ×10⁻⁴ ± 2.9 × 10⁻⁵ TB-A H_F68A 7.8 nd nd TB-A H_F68V/F70L 7.8 nd nd TB-AH_F70L 7.8 nd nd

Yet another relevant feature of the antibodies or antibody derivativesof the present invention is their high stability. Protein stability ofTB-A, TB-A H_M48L/F68I (SEQ ID NO: 31), TB-A Linker_G2R H_F68L (SEQ IDNO:33), TB-A H_K43R/F68I (SEQ ID NO:34) and TB-A H_F68L (SEQ ID NO:35)was assessed by determining the temperature for onset of unfolding bycircular dichroism and light scattering at both 218 and 292 nm (Tab.II). In this experiment TB-A started to unfold at a temperature of 53°C. whereas its derivatives TB-A H_M48L/F68I (SEQ ID NO:31), TB-ALinker_G2R H_F68L (SEQ ID NO:33), TB-A H_K43R/F68I (SEQ ID NO:34) andTB-A H_F68L (SEQ ID NO:35) showed increased thermal stability (56 and58° C.). All test proteins showed irreversible denaturation andprecipitated upon unfolding, making it impossible to determine themelting temperature. In order to determine midpoint of transition in areversible process, unfolding was induced with guanidine hydrochloride(GdnHCl), to keep the unfolded proteins in solution. In this approachfluorescence emission maxima where determined in by fluorimetry tofollow unfolding. In this set-up, TB-A showed again good stability witha midpoint of transition at 2.07 M GdnHCl. In line with the results fromthermal unfolding the derivatives TB-A Linker_G2R H_F68L (SEQ ID NO:33)and TB-A H_K43R/F68I (SEQ ID NO:34) showed increased stability asdisplayed with higher midpoints of transition, 2.33 and 2.3 M GdnHCl,respectively.

TABLE II Stability features of TB-A derivatives Onset of [GdnHCl] atdenaturation midpoint of SEQ [° C.] transition TB-A 53 2.07M TB-AH_M48L/F68I 58 nd TB-A L_V83E H_V79A nd nd TB-A Linker_G2R H_F68L 582.33M TB-A-QC15.2 56 2.30M TB-A-QC23.2 58 nd TB-A-H_F68A nd nd TB-AH_F68V/F70L nd nd TB-A H_F70L nd nd

The stability of TB-A in human serum, human urine, pig vitreous bodyfluid and pig anterior chamber fluid was assayed by measuring TNFαbinding activity of TB-A after incubation for 3 days at 37° C. in therespective body fluid or in assay buffer (TBSTM) as a positive control.TB-A was diluted in body fluids to a final concentration of 10 μM. Afterthe incubation period dilution series of the samples were assayed byELISA in order to determine the binding constant K_(d) of TB-A (FIG.11). When comparing body fluid samples with the positive control TBSTMsamples, a shift of the K_(d) towards higher concentrations wouldindicate a decrease of active protein during the incubation period. Inour experiments, however, no such shift was detectable, indicating thatthe amount of fully active TB-A remained constant in every body fluidassay due to a high stability of the antibody.

Experiment 3 Binding Features of Humanised Antibody Derivatives

The binding properties of all humanised scFv variants were tested inELISA on recombinant human TNFα. The dissociation constants (K_(d)) forall variants lay within a range of 0.8 to more than 10,000 nM. Thereseems to exist a reverse correlation between the grade of homology tothe human acceptor framework and the affinity of the respective binder(FIG. 4A). Nevertheless, some TB-A variants containing mutations towardsthe TB-B sequence show affinity levels towards human TNFα that arecomparable to that of TB-A. FIG. 4B shows two expression yield-improvedderivatives of TB-A (compare FIG. 3A) that exhibit similar affinities asTB-A when compared in ELISA.

TB-A represents a good compromise between the apparent trade-off ofexpression yield and affinity. In terms of affinity, no significantdifference between the single-chain and the Fab fragment format of TB-Awas detectable (data not shown).

The affinity for TNFα and binding kinetics were also determined for TB-Aby surface plasmon resonance (BIACore), resulting in a dissociationconstant K_(d)=0.8 nM, an off rate of k_(off)=4.4×10⁻⁴ s⁻¹ and a on rateof k_(on)=5×10⁵ s⁻¹M⁻¹.

Experiment 4 L929 Cytotoxicity Assay

The function of antibodies or antibody derivatives in neutralising TNFαin vivo can be tested by measuring inhibition of cytotoxicity of TNFαtowards cultured mouse L929 fibroblasts or alternatively towards humanKYM-1 myelosarcoma cells (Tab. III). The humanised scFv derivatives ofDi62 display different efficacies in the L929 assay as shown in FIG. 5B.Some scFv derivatives show IC50 (inhibitory concentration to achieve 50%inhibition) values in the range of 5 ng/ml, whereas others had no effectin the L929 assays. ELISA data and results from the L929 assay do notalways correlate. KYM-1 data and L929 results, however, correlatednicely with the only difference that much higher concentrations ofrecombinant human TNF and consequently also of the antagonist whererequired to see an effect. KYM-1 was, therefore, mainly used to confirmL929 results. For direct comparison of test proteins, potency isexpressed as a relative value normalized to TB-A (EC₅₀X/EC₅₀TB-A). Ingeneral, however, IC50 values become again higher the closer thesequence of a binder is to the human acceptor framework (FW2.3). FIG. 5compares the potency of different derivatives of TB-A to block TNFαinduced cytotoxicity towards mouse L929 fibroblast cells. Absorption at450 nm correlates with cell survival.

TB-A and the anti-hTNFα IgG Infliximab® show a similar IC₅₀ value in theL929 assay, whereas the potency of TB-wt to block human TNFα inducedcytotoxicity is significantly lower (FIG. 5A). When TB-A derivatives arecompared with TB-A for their potential to block TNFα inducedcytotoxicity, most of these derivatives except TB-H43 have a reducedefficacy in the L929 (FIG. 5B).

TABLE III Functional properties of TB-A derivatives Relative potency:EC₅₀X/EC₅₀TB-A SEQ L929 cells KYM-1 cells TB-A 1.0 1.0 TB-A H_M48L/F68I1.1 1.6 TB-A L V83E H V79A Nd nd TB-A Linker_G2R H_F68L 0.8 1.3TB-A-QC15.2 1.37 1.5 TB-A-QC23.2 1.32 1.5 TB-A-H_F68A 1.14 nd TB-AH_F68V/F70L 1.28 nd TB-A H_F70L 2.7 nd

In line with the ELISA data, there is no significant difference in theability to block TNFα-induced cytotoxicity between the scFv and the Fabformat of TB-A (FIG. 5C). The IC₅₀ value of the TB-A Fab format liesabout a factor of two above the IC₅₀ value of the TB-A scFv format (FIG.5C), most probably as a result of the higher molecular mass of the Fabfragment.

Experiment 5 Animal Experiments with Anti-TNFα Antibody Derivatives

5.1. Description of Experiment

In order to test for the efficacy of ESBATech's anti-TNFα antibodyderivatives (scFv and Fab) in functionally neutralising human TNFαbioactivity in an in vivo situation, a recently published rat model foracute monoarthritis was used. This model has extensively been describedby Bolon and colleagues (see Bolon et al. (2004), Vet. Pathol.41:235-243). Briefly, in this animal arthritis model, human TNFα isinjected intra-articularly into the knee joint of male Lewis rats.Injection of human TNFα leads to an acute, self-limiting monoarthritisin the injected joint. Arthritis can be quantified by measurement ofjoint swelling and histological scoring. Consequently, bioactivity ofrespective TNFα antagonists can be quantified by reduction ofTNFα-induced joint swelling and/or reduction of histological parametersof inflammation.

5.2. Materials and Methods

Experimental Design

The current studies were designed to examine the respective potential ofa representative scFv antibody and a representative Fab antibody of theseries described above with the marketed antibody Infliximab (Remicade®)to inhibit bioactivity of human TNFα in an appropriate animal arthritismodel. Bolon and colleagues had shown before that intra-articularapplication of 10 microgram of recombinant human TNFα into the rat kneejoint provokes an acute, self-limiting monoarthritis that can bequantified by standard macroscopic and microscopic analysis. Thus thisanimal model served as ideal system to assess the therapeutic effect oflocally applied antibody derivatives. Two experiments were completed inseries (Tab. IV and V). 1) A basic efficacy study assessing the overallpotential of the antibodies to block human TNFα-induced monoarthritis;and 2) A dose response study assessing the relative efficacy of theantibody derivatives among each other. Both, cytokines and antibodyderivatives were given once by separate injections, as described below.The cytokine dose used was based on the publication by Bolon andcolleagues, whereas the range of doses of the antibody derivatives wasbased on available cell culture data and educated guessing. Theexperiments were conducted in accordance with general animal careguidelines.

Animals and Husbandry

Young, adult male Lewis rats (6-7 weeks and 175-200 g) were randomlyassigned to treatment groups (n=3/cohort) and housed according to Bolonet al. (2004), Vet. Pathol. 41:235-243.

Cytokine and Antibody Instillation

Anaesthesia and cytokine injections were performed as described by Bolonand colleagues. In order not to exceed a total intraarticularly injectedvolume of 50 microliter, cytokines and antibody derivatives wereinstilled in two separate intra-articular injections whereby the 10micrograms of recombinant human TNFα was injected in 10 microliter offilter-sterilised phosphate-buffered saline (PBS) and the respectivedose of the respective antibody was injected in 40 microliter offilter-sterilised phosphate-buffered saline. Animals treated withintraperitoneally applied Infliximab/Remicade® were i.p. injected withthe antibody derivatives 3 hours prior to intra articular injection ofthe human TNFα

In all animals treated with intraarticularly applied antibodytreatments, the respective antibody dose was injected 5 minutes prior toinjection of the human TNFα. Control animals were injected with 10microliter of PBS without human TNFα

Infliximab/Remicade® used in the experiments was purchased at anofficial Swiss pharmacy. Anti-human TNFα specific scFv and Fab (TB-A)antibodies as well as a naïve scFv antibody framework used as unspecificcontrol antibody in the dose response experiment were expressed in E.coli and purified by standard methods. Endotoxin contamination was heldbelow 10 EU per milligram protein in all preparations because thelipopolysaccharide component is a potent inducer of TNFα.

Recombinant human TNFα was purchased from PeproTech EC Ltd.

Measurement of Joint Diameter

Immediately before the injection of the respective, intraperitoneally orintraarticularly applied antibody derivative, or, in case of the controlanimals, before the injection of PBS or TNFα, the diameter of the kneejoint to be injected was determined by means of a standard calliper. 48hours after injection of TNFα (or PBS in control animals) andimmediately before euthanisation of the animals, the diameter of theinjected knee joint was determined again and joint swelling wascalculated by subtracting the value of the second diameter measurementfrom the value of the first diameter measurement (FIGS. 6 and 9).

Tissue Processing

48 hours after injection of TNFα (or PBS in case of control animals)animals were euthanised. At necropsy, injected knees were separated fromthe foot and thigh, fixed intact by immersion in 70% ethanol andproceeded for standard hematoxylin and eosin (HE) staining, as describedby Bolon and colleagues.

Morphologic Analysis

Histological scoring analysis for measurement of joint inflammation wasperformed as described by Bolon and colleagues. Histopathologicalscoring criteria for assessment of joint inflammation were appliedaccording to Bolon and colleagues (FIGS. 7, 8 and 10).

5.3. Results

In a first set of experiments a representative intraarticularly appliedESBATech scFv antibody, TB-A, and the corresponding intraarticularlyapplied ESBATech Fab antibody were compared for their ability to blockinduction of the acute monoarthritis with intraarticularly andintraperitoneally applied Infliximab/Remicade® according to Table IV:

TABLE IV Injection scheme experiment 1 GROUP TNFα (

 g) in PBS INHIBITOR DOSE (

g)  1 (n = 3) 0 none  2 (n = 3) 10 none  3 (n = 3) 0 TB-A scFv 180  4 (n= 3) 10 TB-A scFv 180  5 (n = 3) 0 TB-A 450   Fab antibody  6 (n = 3) 10TB-A 450   Fab antibody  7 (n = 3) 10 TB-A 180   Fab antibody  8 (n = 3)0 Infliximab (i.a.) 450  9 (n = 3) 10 Infliximab (i.a.) 450 10 (n = 3)10 Infliximab (i.a.) 180 11 (n = 3) 10 Infliximab (i.p.) 450 12 (n = 3)10 Infliximab (i.p.) 180

The results obtained regarding treatment effects on change of kneediameter (as an indicator of effects on TNFα-induced joint swelling) arerepresented in FIG. 6. All antibodies completely blocked TNFα-inducedjoint swelling.

For evaluation of treatment effects on joint inflammation, histologicalscoring of HE-stained tissue slides was performed. Joint inflammationwas scored by the following criteria (see FIG. 7 for representativescoring examples):

-   Score 0: normal-   Score 1: Mild thickening of synovial lining-   Score 2: Thickening of synovial lining and mild inflammation of the    sublining-   Score 3: Thickening of synovial lining and moderate inflammation of    the sublining

The results obtained regarding treatment effects on histopathologicalinflammation scores are shown in FIG. 8.

Comparable effects of all treatment on histopathological inflammationscores were observed.

In a second set of experiments, the relative dose response to theassessed antibody derivatives was compared. The representativeintraarticularly applied ESBATech scFv antibody TB-A and thecorresponding intraarticularly applied Fab antibody of experiment 1 werecompared with intraarticularly and intraperitoneally appliedInfliximab/Remicade® and an unrelated scFv antibody lacking any bindingactivity to human TNFα over a broader and different dose range ascompared with experiment 1, according to Table V.

TABLE V Injection scheme experiment 2 GROUP TNFα (

 g) in PBS INHIBITOR DOSE (

g)  1 (n = 3)  0 none  2 (n = 3) 10 none  3 (n = 3) 10 Unrelated scFv180   antibody  4 (n = 3) 10 TB-A scFv antibody 156  5(n = 3) 10 TB-AscFv antibody 45  6 (n = 3) 10 TB-A scFv antibody 11  7 (n = 3) 10 TB-AFab antibody 156  8 (n = 3) 10 TB-A Fab antibody 45  9 (n = 3) 10 TB-AFab antibody 11 10 (n = 3) 10 Infliximab (i.a.) 156 11 (n = 3) 10Infliximab (i.a.) 45 12 (n = 3) 10 Infliximab (i.a.) 11 13 (n = 3) 10Infliximab (i.p.) 156 14 (n = 3) 10 Infliximab (i.p.) 45 15 (n = 3) 10Infliximab (i.p.) 11

The results obtained regarding treatment effects on change of kneediameter (as an indicator of effects on TNFα-induced joint swelling) areshown in FIG. 9.

The results obtained regarding treatment effects on histopathologicalinflammation scores are presented in FIG. 10.

In summary, both the representative ESBATech anti-TNFα scFv and therepresentative ESBATech anti-TNFα Fab antibody were highly efficient inblocking human TNFα-induced monoarthritis upon local (intraarticular)administration.

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpracticed within the scope of the following claims.

The invention claimed is:
 1. A method for the production of an antibodywhich specifically binds TNFα, said antibody comprising a light chainvariable domain (VL) comprising the sequence of SEQ ID NO:1 and a heavychain variable domain (VH) comprising the sequence of SEQ ID NO:2, or anantigen-binding derivative thereof, wherein said derivative has atmaximum up to five amino acid changes as compared to SEQ ID NO: 1 and/orat maximum up to nine amino acid changes as compared to SEQ ID NO: 2,wherein said changes occur at amino acid positions in framework regionsof said VL comprising the sequence of SEQ ID NO: 1 and said VHcomprising the sequence of SEQ ID NO: 2, and wherein said derivativedoes not comprise the entire sequences of SEQ ID NO: 3 and SEQ ID NO: 4,the method comprising the steps of: culturing a host cell underconditions that allow the synthesis of said antibody, and recovering itfrom said culture.
 2. The method of claim 1, wherein said host cell is aprokaryotic cell.
 3. The method of claim 2, wherein said host cell is E.coli.
 4. The method of claim 1, wherein said host cell is an eukaryoticcell.
 5. The method of claim 4, wherein said eukaryotic cell is a yeast,plant, insect or mammalian cell.
 6. The method of claim 1, wherein theup to 5 changes of VL in the antigen-binding derivative are at any ofthe positions 4, 46, 65, 67, 70, and 83 and the up to 9 changes of VH inthe antigen-binding derivative are at any of the positions 11, 16, 28,43, 68, 70, 71, 72, 73, 76, 77, 93 and
 112. 7. The method of claim 1,wherein the up to 5 changes of VL in the antigen-binding derivative areat any of the positions 4, 46, 65, 67, 70, and 83 and the up to 9changes of VH in the antigen-binding derivative are at any of thepositions 11, 16, 28, 43, 48, 68, 70, 71, 72, 73, 76, 77, 79, 93 and112.
 8. The method of claim 1, in which at least one of the changes insaid antigen-binding derivative leads to an amino acid present in SEQ IDNO:3 at a corresponding position in SEQ ID NO: 1 for VL and/or leads toan amino acid present in SEQ ID NO:4 at a corresponding position in SEQID NO: 2 for VH, wherein said derivative does not comprise the entiresequences of SEQ ID NO: 3 and SEQ ID NO:
 4. 9. The method of claim 1,wherein the VL domain of said antibody or antigen-binding derivativecomprises the sequence of SEQ ID NO:
 1. 10. The method of claim 9comprising the VH domain of said antibody or antigen-binding derivativeof the sequence of SEQ ID NO:2.
 11. The method of claim 9, wherein saidantigen-binding derivative comprises a VH domain derived from thesequence of SEQ ID NO:2, wherein F68 is changed to A, L, I, or V. 12.The method of claim 1, wherein said antigen-binding derivative comprisesthe VL domain of the sequence of SEQ ID NO:11, and the VH domain of thesequence of SEQ ID NO:4.
 13. The method according to claim 1, whereinsaid antigen-binding derivative is an scFv antibody wherein the VL andVH domains are connected by a linker.
 14. The method according to claim13, wherein said scFv antibody comprises a VL-linker-VH sequencearrangement.
 15. The method according to claim 13, wherein the linkerhas the sequence of SEQ ID NO:10 or is derived from said sequence. 16.The method of claim 15, wherein at least one G of said linker is changedto a more polar or charged amino acid.
 17. The method of claim 16,wherein the linker has the sequence of SEQ ID NO:39.
 18. The methodaccording to claim 1, wherein said antigen-binding derivative is a Fabfragment wherein the VL domain is fused to the constant region of ahuman Ig kappa chain, the VH domain is fused to the CH1 domain of ahuman IgG, and the two fusion polypeptides are connected by aninter-chain disulfide bridge.
 19. The method according to claim 13 or18, wherein said scFv antibody or Fab fragment is labeled or chemicallymodified.